Areca nut actions on fibroblasts are enhanced by epithelial mesenchymal interaction via TGF-β.

<p>To study the epithelial mesenchymal interaction, confluent cultures of HaCaT cells were serum starved for 24 hours followed by 10 μM ALK5 inhibitor (TGFβRI inhibitor, SB 431542, Sigma-Aldrich, USA) treatment 2 hours prior to areca nut treatment (5 μg/ml). Meanwhile hGF cells were serum deprived for 24 hours such that the treatment time point coincided with completion of 48 hour treatment on HaCaT cells. At this time point, the condition medium of areca nut (with or without ALK5 inhibitor; SB 431542) treated HaCaT cells was transferred to hGF cells and simultaneously direct treatment of areca nut with or without ALK5 inhibitor; SB 431542 was also performed and both were maintained for 48 hours and gene expression was studied by qPCR. The bar diagrams represent regulation of αSMA/ACTA2, γSMA/ACTG2, THBS1, TGM2, TMEPAI, TGFBI, CTGF, PLOD2, BMP1, LIMK1, LOXL3 and EDN1 in hGF cells upon treatment with 1- untreated; 2- areca nut, 3- ALK5 inhibitor(SB 431542), 4- areca nut with ALK5 inhibitor (SB 431542) (white bars), 5- condition media of untreated HaCaT cells, 6- condition media of areca nut treated HaCaT cells, 7- condition media of ALK5 inhibitor (SB 431542) treated HaCaT cells and 8- condition media of areca nut with ALK5 inhibitor (SB 431542)treated HaCaT cells (black bars). P values <0.0001, <0.001, <0.01 are depicted as ***, ** and * respectively.